Transforming Growth Factor-β Induces Secretion of Activated ADAMTS-2
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m300767200
ISSN1083-351X
AutoresWei-Man Wang, Seungbok Lee, Barry M. Steiglitz, Ian C. Scott, Carter C. Lebares, M. Leah Allen, Mitchell C. Brenner, Kazuhiko Takahara, Daniel S. Greenspan,
Tópico(s)Platelet Disorders and Treatments
ResumoThe metalloproteinase ADAMTS-2 has procollagen I N-proteinase activity capable of cleaving procollagens I and II N-propeptides in vitro, whereas mutations in the ADAMTS-2 gene in dermatosparaxis and Ehlers-Danlos syndrome VIIC show this enzyme to be responsible in vivo for most biosynthetic processing of procollagen I N-propeptides in skin. Yet despite its important role in the regulation of collagen deposition, information regarding regulation and substrate specificity of ADAMTS-2 has remained sparse. Here we demonstrate that ADAMTS-2 can, like the procollagen C-proteinases, be regulated by transforming growth factor-β1 (TGF-β1), with implications for mechanisms whereby this growth factor effects net increases in formation of extracellular matrix. TGF-β1 induced ADAMTS-2 mRNA ∼8-fold in MG-63 osteosarcoma cells in a dose- and time-dependent, cycloheximide-inhibitable manner, which appeared to operate at the transcriptional level. Secreted ADAMTS-2 protein induced by TGF-β1 was 132 kDa and was identical in size to the fully processed, active form of the protease. Biosynthetic processing of ADAMTS-2 to yield the 132-kDa form is shown to be a two-step process involving sequential cleavage by furin-like convertases at two sites. Surprisingly, purified recombinant ADAMTS-2 is shown to cleave procollagen III N-propeptides as effectively as those of procollagens I and II, whereas processing of procollagen III is shown to be decreased in Ehlers-Danlos VIIC. Thus, the dogma that procollagen I and procollagen III N-proteinase activities are provided by separate enzymes appears to be false, whereas the phenotypes of dermatosparaxis and Ehlers-Danlos VIIC may arise from defects in both type I and type III collagen biosynthesis. The metalloproteinase ADAMTS-2 has procollagen I N-proteinase activity capable of cleaving procollagens I and II N-propeptides in vitro, whereas mutations in the ADAMTS-2 gene in dermatosparaxis and Ehlers-Danlos syndrome VIIC show this enzyme to be responsible in vivo for most biosynthetic processing of procollagen I N-propeptides in skin. Yet despite its important role in the regulation of collagen deposition, information regarding regulation and substrate specificity of ADAMTS-2 has remained sparse. Here we demonstrate that ADAMTS-2 can, like the procollagen C-proteinases, be regulated by transforming growth factor-β1 (TGF-β1), with implications for mechanisms whereby this growth factor effects net increases in formation of extracellular matrix. TGF-β1 induced ADAMTS-2 mRNA ∼8-fold in MG-63 osteosarcoma cells in a dose- and time-dependent, cycloheximide-inhibitable manner, which appeared to operate at the transcriptional level. Secreted ADAMTS-2 protein induced by TGF-β1 was 132 kDa and was identical in size to the fully processed, active form of the protease. Biosynthetic processing of ADAMTS-2 to yield the 132-kDa form is shown to be a two-step process involving sequential cleavage by furin-like convertases at two sites. Surprisingly, purified recombinant ADAMTS-2 is shown to cleave procollagen III N-propeptides as effectively as those of procollagens I and II, whereas processing of procollagen III is shown to be decreased in Ehlers-Danlos VIIC. Thus, the dogma that procollagen I and procollagen III N-proteinase activities are provided by separate enzymes appears to be false, whereas the phenotypes of dermatosparaxis and Ehlers-Danlos VIIC may arise from defects in both type I and type III collagen biosynthesis. Collagen types I–III, the major fibrous constituents of vertebrate extracellular matrix (ECM), 1The abbreviations used are: ECM, extracellular matrix; N-propeptide, N-terminal propeptide; C-propeptide, C-terminal propeptide; pCP, procollagen C-proteinase; pNP, procollagen N-proteinase; EDSVIIC, Ehlers-Danlos Syndrome type VIIC; ADAMTS, a disintegrin and metalloproteinase with thrombospondin type I motifs; TGF-β1, transforming growth factor-β1; BMP-1, bone morphogenetic protein-1; mTLD, mammalian Tolloid; mTLL-1, mammalian Tolloid-like 1; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F. are synthesized as procollagen precursors with N- and C-propeptides that are proteolytically removed to produce mature monomers capable of forming fibrils (1van der Rest M. Garrone R. FASEB J. 1991; 5: 2814-2823Crossref PubMed Scopus (998) Google Scholar, 2Hulmes D.J.S. 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Byers P.H. Lapière C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; 65: 308-317Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 22Colige A. Li S.-W. Sieron A.L. Nusgens B.V. Prockop D.J. Lapière C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2374-2379Crossref PubMed Scopus (158) Google Scholar). Analysis of sequences demonstrated that mutations in the pNP gene lead to the recessively inherited human disorder Ehlers-Danlos syndrome type VIIC (EDSVIIC) and the analogous disease dermatosparaxis in cattle (21Colige A. Sieron A.L. Li S.-W. Schwarze U. Petty E. Wertelecki W. Wilcox W. Krakow D. Cohn D.H. Reardon W. Byers P.H. Lapière C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; 65: 308-317Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), both of which are marked by extreme fragility of the skin and accumulation in the skin of processing intermediates in which the C- but not the N-propeptides of type I procollagen have been removed (23Lenaers A. Ansay M. Nusgens B. Lapière C.M. Eur. J. Biochem. 1971; 23: 533-543Crossref PubMed Scopus (200) Google Scholar, 24Nusgens B.V. Verellen-Dumoulin G. Hermans-Le T. De Paepe A. Nuytinck L. Piérard G.E. Lapière C.M. Nat. Genet. 1992; 1: 214-217Crossref PubMed Scopus (102) Google Scholar, 25Smith L.T. Wertelecki W. Milstone L.M. Petty E.M. Seashore M.R. Braverman I.M. Jenkins T.G. Byers P.H. Am. J. Hum. Genet. 1992; 51: 235-244PubMed Google Scholar). Analysis of sequences also showed pNP to belong to the recently described ADAMTS (ADisintegrin And Metalloproteinase with ThromboSpondin motifs) family of metalloproteinases (26Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) and led to designation of pNPI as ADAMTS-2. At present there are 19 reported vertebrate ADAMTS family members (27Cal S. Obaya A.J. Llamazares M. Garabaya C. Quesada V. Lopez-Otin C. Gene (Amst.). 2002; 283: 49-62Crossref PubMed Scopus (224) Google Scholar, 28Somerville R.P. Longpre J.M. Engle J.M. Jungers K.A. Ross M. Evanko S. Wight T.N. Leduc R. Apte S.S. J. Biol. Chem. 2003; 278: 9503-9513Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), which share a common domain structure. They resemble the ADAMs family of proteases in having pro-, adamalysin/reprolysin-like metalloprotease, disintegrin-like and cysteine-rich protein domains but differ in that they lack epidermal growth factor-like domains, and unlike many ADAMs proteases, they lack transmembrane domains (26Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 29Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Instead, ADAMTS proteases contain variable numbers of thrombospondin type I-like repeats (26Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 29Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) which, at least in some family members, appear to be involved in binding to components of the ECM (30Kuno K. Matsushima K. J. Biol. Chem. 1998; 273: 13912-13917Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). There are indications that ADAMTS proteases play a broad range of roles in development, reproduction, disease, and homeostasis. Aside from the role of ADAMTS-2 in biosynthetic processing of procollagens I and II, the phenotype of Adamts2-null mice suggests a role in male fertility as well (31Li S.-W. Arita M. Fertala A. Bao Y. Kopen G.C. Långsjö T.K. Hyttinen M.M. Helminen H.J. Prockop D.J. Biochem. J. 2001; 355: 271-278Crossref PubMed Scopus (102) Google Scholar). ADAMTS-1, -4, and -5/11 appear to constitute a subset of highly homologous ADAMTS family members with aggrecanase activity, important to homeostasis of cartilage and etiology of the arthritides (32Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (625) Google Scholar, 33Abbaszade I. Liu R.-Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 34Kuno K. Okada Y. Kawashima H. Nakamura H. Miyasaka M. Ohno H. Matsushima K. FEBS Lett. 2000; 478: 241-245Crossref PubMed Scopus (236) Google Scholar). ADAMTS-1 was originally identified as a gene product induced by inflammation and associated with cachexia (26Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). The phenotype of Adamts1-null mice also suggests roles for ADAMTS-1 in growth, organogenesis, and female fertility (35Shindo T. Kurihara H. Kuno K. Yokoyama H. Wada T. Kurihara Y. Imai T. Wang Y. Ogata M. Nishimatsu H. Moriyama N. Oh-hashi Y. Morita H. Ishikawa T. Nagai R. Yazaki Y. Matsushima K. J. Clin. Invest. 2000; 105: 1345-1352Crossref PubMed Scopus (275) Google Scholar), whereas both ADAMTS-1 and ADAMTS-8 have been shown to have anti-angiogenic activity (36Vázquez F. Hastings G. Ortega M.-A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Roles for ADAMTS-like proteins in fertility and organogenesis in a broad spectrum of species are suggested by the finding that the gon-1 gene, which encodes an ADAMTS-like product, is essential for gonadal morphogenesis in Caenorhabditis elegans (37Blelloch R. Kimble J. Nature. 1999; 399: 586-590Crossref PubMed Scopus (166) Google Scholar). Mutations in the gene for ADAMTS-13 have been shown to be causal for thrombotic thrombocytopenic purpura, perhaps due to its demonstrated ability to process von Willebrand factor, showing ADAMTS-13 to have an important role in human vascular homeostasis (38Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemlenlak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sablo H. Bouhassira E.E. Upshaw Jr., J.D. Ginsburg D. Tsai H.-M. Nature. 2001; 413: 488-494Crossref PubMed Scopus (1467) Google Scholar). Despite the seeming importance of ADAMTS proteases in many biological processes, there has been little characterization of the regulation of their expression and activity. In a previous study of the regulation of procollagen C-proteinase activity and expression of BMP-1 and mTLD, by transforming growth factor-β1 (TGF-β1), we noted elevation of both pCP and pNPI activities in MG-63 osteosarcoma cells treated with TGF-β1 (39Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In the present study, we show that TGF-β1 induces expression of ADAMTS-2 mRNA, apparently at the transcriptional level, resulting in expression of fully processed and active 132-kDa ADAMTS-2 protein from MG-63 cells. Processing of the ADAMTS-2 pro-domain is shown to be via a two-step process involving sequential cleavage by furin-like proprotein convertases at two consensus sites. Surprisingly, purified ADAMTS-2 protein is shown to have pNP activity not only against procollagens I and II but against procollagen III as well, whereas processing of the procollagen III N-propeptide is shown to be decreased in EDSVIIC fibroblast cultures. These latter two findings indicate that the distinction that has been made between pNPI and pNPIII is a false one. Implications of the various data are discussed. RNA Analysis—To generate probes for analysis of human ADAMTS-2 RNA, ADAMTS-2 sequences were PCR-amplified from human dermal fibroblast cDNA (Clontech) using oligonucleotide primers 5′-TTTGGCCGAGACCTGCACCTGC-3′ (forward) and 5′-TGACAGGAGCATAGCCTTGCATGC-3′ (reverse), corresponding to nucleotides 346–367 and 1147 to 1124, respectively, of the published ADAMTS-2 sequence (GenBank™ accession number AJ003125, see Ref. 21Colige A. Sieron A.L. Li S.-W. Schwarze U. Petty E. Wertelecki W. Wilcox W. Krakow D. Cohn D.H. Reardon W. Byers P.H. Lapière C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; 65: 308-317Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The resulting PCR product was used to screen a λgt10 human placenta cDNA library (Clontech), yielding one positive clone with a 1231-bp insert extending from nucleotides 400 to 1630 that was subcloned into the EcoRI site of pBluescript II KS+ (Stratagene). This insert was employed as a Northern blot probe. Restriction of the described construct with PstI, which cleaves after ADAMTS-2 nucleotide 786 and within the pBluescript polylinker, followed by religation of the plasmid, yielded a template that, upon linearization with EcoRI and transcription with T7 polymerase, yielded a 387-base riboprobe used for RNase protection assays. Poly(A+) RNA was prepared from cultured MG-63 human osteosarcoma cells as described previously (39Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Unless otherwise noted in the text, TGF-β1 treatment of cells harvested for RNA was at 2 ng/ml for 24 h. To test stability of ADAMTS-2 mRNA, just confluent MG-63 cells, untreated or treated with TGF-β1 as described above, were then treated with 10 μg/ml actinomycin D for varying times in Dulbecco's modified Eagle's medium (DMEM) containing 0.1% fetal bovine serum (FBS) containing or lacking 2 ng/ml TGF-β. Stability was determined by RNase protection assays. RNase protection and Northern blot assays were performed as described by Lee et al. (39Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Production of Recombinant Protein—For expression of recombinant ADAMTS-2 in 293-EBNA cells, a human ADAMTS-2 cDNA insert extending from nts 112 to 3633 of the published sequence (21Colige A. Sieron A.L. Li S.-W. Schwarze U. Petty E. Wertelecki W. Wilcox W. Krakow D. Cohn D.H. Reardon W. Byers P.H. Lapière C.M. Prockop D.J. Nusgens B.V. Am. J. Hum. Genet. 1999; 65: 308-317Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), corresponding to the full-length protein minus signal peptide sequences and with PCR addition of sequences encoding DYKDDDDK-Stop to the 3′-end of the insert, was ligated between the NheI and BamHI sites of episomal expression vector pCEP-Pu/BM40s (40Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar). Coding was thus for full-length human ADAMTS-2 differing from the native protein only in replacement of the native signal peptide by BM40 signal peptide sequences, to enhance secretion, and by addition of a C-terminal FLAG tag. Experiments in Fig. 7 were performed with recombinant ADAMTS-2 from a clonal line of transfected 293 cells constitutively expressing ADAMTS-2 and prepared toward the end of the study. To prepare this clonal line, in-frame BM40s/ADAMTS-2/FLAG coding sequences were excised from pCEP-Pu with AflII and BamHI and inserted between corresponding sites of expression vector pcDNA3.1 (Invitrogen), which was transfected into 293 cells. 293 cells were grown in growth medium consisting of DMEM supplemented with 1 mm l-glutamine and 10% FBS, whereas 293-EBNA cells were grown in the same growth medium supplemented with 250 μg/ml G418 (Invitrogen). Transfection of both types of cells was at 90% confluence with 10 μg of expression vector per 100-mm culture dish, using LipofectAMINE (Invitrogen). After 36 h, transfected 293-EBNA cells were selected, and surviving cells were allowed to grow to confluent mass cultures in growth medium containing 5 μg/ml puromycin (Sigma) and 250 μg/ml G418. After 72 h, transfected 293 cells were selected in growth medium containing 500 μg/ml G418 (Invitrogen), and surviving colonies of cells were ring cloned. Culture media of 36 clones were analyzed by immunoblot, and the clone expressing the highest levels of ADAMTS-2 was used for the studies of Fig. 7. To prepare purified recombinant ADAMTS-2, mass cultures of transfected 293-EBNA cells were grown to confluence, washed twice with phosphate-buffered saline (PBS), and switched to serum-free DMEM containing 40 μg/ml soybean trypsin inhibitor (Sigma) with or without 5 μg/ml soluble heparin (Sigma). Conditioned media were harvested 24 h later, and protease inhibitors were added to final concentrations of 0.4 mm phenylmethylsulfonyl fluoride and 10 μg/ml leupeptin. Harvested media were centrifuged to remove debris, and supernatants were stored at -70 °C. FLAG-tagged ADAMTS-2 in media was bound to 1 ml of FLAG M2 matrix, and the matrix was washed with 10 ml of binding buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.4), using the supplier's protocols (Sigma). ADAMTS-2 was eluted with 1 mg/ml FLAG peptide (Sigma) in binding buffer, and fractions were collected and analyzed by Western blot. Purified full-length recombinant human ADAMTS-1, ADAMTS-4, and ADAMTS-5 were provided by Wyeth Research, Cambridge, MA. Preparation of Cell Lysate and ECM—For the experiments of Fig. 7, confluent cells were rinsed twice with PBS, detached in PBS containing 5 mm EDTA, pelleted by centrifugation, and lysed in 1× SDS-PAGE loading buffer. ECM remaining on dishes was rinsed twice with PBS and then scraped into 1× SDS-PAGE loading buffer. Treatment of cells in serum-free growth media with 20 μm of furin inhibitor decanoyl-RVKR-chloromethyl ketone (Bachem) was as described previously (41Unsöld C. Pappano W.N. Imamura Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 2002; 277: 5596-5602Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Induction of Secreted ADAMTS-2 by TGF-β—MG-63 cells at 70% confluence were trypsinized and replated in low salt, low cysteine, low sulfate DMEM containing 10% dialyzed FBS (Hyclone), 4 mm l-glutamine, and 30 mm sodium chlorate. After 48 h, cells were trypsinized again and replated in the same type of chlorate-containing media. The next day, cells were switched to serum-free, chlorate-containing DMEM with 2 ng/ml TGF-β1 (R & D Systems) and 40 μg/ml soybean trypsin inhibitor, and conditioned media were harvested 48 h later. Cell layers were then treated 2 h with 0.006 IU/ml heparitinase (Seikagaku America) in serum-free DMEM at 37 °C. These media were removed and pooled with conditioned media from the previous step, and pooled media were incubated 20 h with heparin-Sepharose beads (Amersham Biosciences) at 4 °C. Beads were centrifuged 1 min at 1,000 × g at 4 °C and washed three times with PBS, and protein was eluted by boiling beads for 5 min in 30 μl of 4× SDS-PAGE loading buffer. Immunoblots—Samples subjected to SDS-PAGE were transferred to Immobilon-P membranes (Millipore) as described (39Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Blots were blocked 1 h with 2% bovine serum albumin in T-PBS (PBS, 0.05% Tween 20) and incubated overnight with primary antibody diluted 1:5000 in the same solution. Blots were washed with T-PBS and blocked again by 2% bovine serum albumin in T-PBS, followed by incubation with 1:5000 diluted secondary antibody. After washing 6 times in T-PBS, blots were incubated 4 min in SuperSignal West Pico substrate (Pierce) and exposed to film. The blot in the right panel of Fig. 9B was washed overnight in T-PBS after transfer, blocked 1 h with 3% bovine serum albumin in T-PBS, incubated overnight with primary antibody diluted 1:2000 in the same solution and, the next day, washed as above. Antibodies 1429, 1435, 1374, and 1442, raised against peptides whose sequences are presented in Fig. 3, were prepared and affinity-purified using techniques described previously (39Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Peptide antibodies directed against sequences in the pro-α1(I) C-telopeptide (LF-67), pro-α1(III) C-propeptide (LF-69), and N-telopeptide (LF-71) have been described previously (42Bernstein E.F. Chen Y.Q. Kopp J.B. Fisher L. Brown D.B. Hahn P.J. Robey F.A. Lakkakorpi J. Uitto J. J. Am. Acad. Dermatol. 1996; 34: 209-218Abstract Full Text PDF PubMed Scopus (179) Google Scholar) and were the kind gifts of Dr. Larry Fisher (NIDCR, National Institutes of Health, Bethesda). Although antibody LF-71 was raised against pro-α1(III) N-telopeptide sequences, it appears to recognize only forms that retain the N-propeptide (i.e. pro-α1(III) and pNα(III) chains) and does not recognize forms from which the N-propeptide has been removed (i.e. pCα1(III) and mature α1(III) chains), perhaps due to conformational changes affecting availability of epitopes 3L. W. Fisher and P. H. Byers, personal communication. (42Bernstein E.F. Chen Y.Q. Kopp J.B. Fisher L. Brown D.B. Hahn P.J. Robey F.A. Lakkakorpi J. Uitto J. J. Am. Acad. Dermatol. 1996; 34: 209-218Abstract Full Text PDF PubMed Scopus (179) Google Scholar).Fig. 3Structural features of ADAMTS-2.Boxes in the schematic of ADAMTS-2 represent the signal peptide (S); the pro- (Pro); protease (Protease); disintegrin-like (Dis); 1st, 2nd, 3rd, and 4th thrombospondin type-1 (Tsp1, 2, 3, and 4); cysteine-rich (Cys); spacer (Spacer); and C-terminal domains (C-term). Placement of the signal peptide cleavage site, between proline residues 29 and 30, was determined using the method of Nielsen et al. (55Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4942) Google Scholar). The dashed region within the protease domain represents the Zn2+-binding active site (Zn2+). The dashed region within the C-terminal domain indicates the site of the relatively conserved PLAC region. Arrows indicate sites of consensus sequences for potential cleavage by furin-like proprotein convertases (56Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Scopus (581) Google Scholar). Closed circles indicate sites of consensus sequences for potential Asn-linked glycosylation. Vertical lines indicate sites corresponding to the N termini of ∼132 and ∼118-kDa forms of ADAMTS-2, as experimentally determined by automated Edman degradation. The cleavage site for production of the ∼132-kDa form was at the more C-terminal of the two furin sites (RARR259/260HAAD), whereas the cleavage sites for the two ∼118-kDa forms were at GFSS402/403AFVV and SYDC465/466LLDD, respectively. Open circles above the schematic indicate the positions of amino acid residues corresponding to peptides used in the production of antibodies 1429, 1435, 1374, and 1442. Below the schematic the ADAMTS-2 (TS-2) amino acid sequences are presented for the peptides used to raise the various antibodies. Alignments show amino acid differences between the ADAMTS-2 peptides and equivalent regions of ADAMTS-3 (TS-3) and ADAMTS-14 (TS-14).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Proteinase Assays—ADAMTS-2, prepared and purified as above, and/or mTLL-1, prepared and purified as described previously (7Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar), were incubated for 20 h with 210 ng of types I–III procollagen in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm CaCl2 at 37 °C. Reactions were stopped by adding 10× SDS-PAGE loading buffer and boiling 5 min. Procollagen substrates, radiolabeled with [2,3-3H]proline, were prepared and purified essentially as described previously (7Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar). Samples were subjected to SDS-PAGE on 5% acrylamide gels, which were treated with EN 3L. W. Fisher and P. H. Byers, personal communication.HANCE (DuPont) and exposed to film. Amino Acid Sequence Analysis—Proteins on 5% acrylamide SDS-PAGE gels were transferred, as above, to Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad). Bands were excised, and N-terminal amino acid sequences were determined by automated Edman deg
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